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THE EMT6 tumour and the methodology of handling used in our laboratory have been previously described (Rockwell, Kallman and Fajardo, 1972; Twentyman and Bleehen, 1974, 1975a). Briefly, following removal of the tumour from the host, it is finely minced with scissors and agitated for 20 min in Hanks' solution containing 0 05%0 trypsin (Gibco Biocult). At the end of this period the resulting suspension is filtered through cotton gauze, centrifuged at 200 g for 5 min, and the cell pellet resuspended in complete Eagle's medium containing 20% calf serum. The resulting suspension, especially if prepared from larger tumours (i.e. > 300 mm3) is heavily contaminated with blood cells and other non-tumour cells. The tumour cells can, however, be counted on a haemocytometer on the basis of size differential, although different workers in our laboratory vary in their counts of the same suspension, because of different subjective limits of size discrimination. Following resuspension of the cell pellet in medium prewarmed to 37 °C, 2-ml aliquots of the suspension were placed into prewarmed 5-cm plastic tissue culture Petri dishes (Sterilin) and placed in a 37 °C incubator in an atmosphere of 950 air + 50 CO2. At intervals thereafter dishes were removed and the medium tipped off. Each dish was then gently rinsed twice with complete medium and twice with a solution of 0OKI% trypsin in phosphate-buffered saline. The dish Accepted 13 September 1976

Separation of clonogenic tumour cells from EMT6 mouse mammary tumours